In order to reliably perform immunoassays, unrestricted access to selected epitopes on target molecules, or analytes, as defined by selected antibodies, are necessary for quantitative determination of the analytes. If two or more different proteins, capable of interacting with each other under physiological conditions, have formed complexes, the component at lower concentration will, depending on the interaction properties and concentrations of the two interactants, partly appear in complexed form with the counterpart. This may prove disadvantageous for quantification particularly of the counterpart in lower concentration as some epitopes used for the assay might be hidden in the complex.
Certain types of proteins may form homo-multimers, e.g. fibrillating proteins, where critical epitopes essential for assaying will not be fully accessible in protein aggregates. If the monomer has a limited number of epitopes this might contribute to underestimation of the monomer protein concentration when multimerization is prone to occur (1).
In order to preserve homeostasis, protein complexes may be formed between active enzymes and their inhibitors in a pre-determined ratio complicating accurate determination of the enzyme. This may lead to certain epitopes being inaccessible in immunoassays and hence an immunoassay may generate inaccurate concentration estimates (2).
Intermittent release of intracellular proteins over longer periods of time due to cell damage of e.g. cardiac cells (3) or tumour cells (4) may generate immune responses against intracellular proteins. These may in a later stage contribute to the formation of immune complexes composed of target molecules and auto-antibodies. Given the amplification properties of the immune system, antibodies may be formed at much higher relative concentrations compared to the target protein leading to formation of immune complexes complicating accurate quantification of target protein.
The above examples represent situations where quantification of target analyte may generate significant deviations from the true target analyte value, often greatly underestimating the true concentration.
Also in biochemical purification processes similar phenomena may occur. In recent years a whole new class of therapeutics, recombinant monoclonal antibodies, has been introduced for treatment of various disorders such as inflammatory diseases, cancer and infection (5). Many of the original therapeutic monoclonal antibodies are purified from cell culture by sequential purification steps employing affinity chromatography, ion exchange chromatography and possibly gel filtration (6). Quite commonly the affinity purification step is based on the interaction between IgG and protein A from Staphylococcus aureus. Protein A immobilized to suitable resins is used as a capturing agent for cell culture containing monoclonal antibodies. This step is very efficient in enriching the desired molecule while contaminants from the cell culture are significantly reduced.
Unfortunately, the ligand used for purification may leach from the resin during the process and end up as an impurity in the purified material. Leaching may occur as a consequence of the dissociation conditions used, for example, proteolytic cleavage of ligand by components from the cell culture, but also the property of resins used, the immobilization chemistry and other aspects related to manufacturing of the affinity resin, as well as the forces involved in the bio-specific interaction between the interactants, may all contribute to ligand leaching to some degree. Irrespective of which specific mechanism is involved, the ligand may contaminate the product being purified on the affinity resin. Depending on the specific biological properties of the impurity ligand, administration of therapeutic proteins purified according to these principles, which may contain biologically active impurities, may induce non-desired side effects, e.g. allergic shock or complement activation, increasing the risk-profile of the treatment.
Native protein A, produced by staphylococci, interacts with immunoglobulins in two principally different manners:                The classical interaction involving the Fc portion of human IgG (7).        The alternative interaction involving immunoglobulins, irrespective of immunoglobulin class (8), that belongs to the VHIII (9) group of the variable domain of the heavy chains.        
Native protein A has five immunoglobulin binding domains (10), each of which can interact independently with IgG portions Fcγ and Fab, respectively. This creates a multitude of interaction possibilities between IgG and protein A, even forming precipitates at equimolar proportions (7). However, it is likely that also under conditions when the proportions of interactants are very dissimilar, heterogeneous complexes will be formed engaging several of the potential interactions in complex formation.
Native protein A has been modified using recombinant technologies (11). One example is when native, staphylococcal protein A or recombinant versions of the same molecule, Fragment Z in multimer version (11), or MabSelect SuRe™ ligand (GE Healthcare Life Sciences, Uppsala, Sweden), a protein A-derived molecule and modified with respect to alkaline tolerance (12) (immobilized on agarose in chromatography medium MabSelect SuRe™) to improve stability upon repeated cleaning-in-place procedures, are used as ligand in the purification process. Thus, during the purification procedure native protein A or its recombinant relatives, respectively, may leach from the resin and form complexes with the eluted IgG once buffer conditions during the continued purification process reach a pH allowing complex formation between protein A and IgG. Attempts to quantify the amount of protein A in relation to IgG expressed as ppm in neutral pH are likely to be severely affected by limited access to relevant epitopes on protein A. This is likely to lead to underestimation of the real concentration of protein A. In order to avoid patient exposure for too high concentrations of leached protein A these levels should be less than 12-14 ppm (13).
Two different principles have been applied to disrupt protein A-IgG complexes to make protein A accessible for quantification:                Heat denaturation of the IgG component present in the sample used for quantification of protein A in the presence of compounds assisting in the denaturation process (14). Protein A is considered to resist denaturation from such treatment. Once the IgG component of the complex is denatured the process will release the protein A moiety for accurate quantification (15).        Acid treatment of sample to dissociate preformed complexes and performance of immunoassay under acid conditions (16; WO 91/10911). Here immune reagents used in the immunoassay must tolerate the selected acid conditions. Optimally the selected pH should, on the one hand, quantitatively dissociate complexes between protein A and IgG (i.e. dissociate protein A from the Fc and/or Fab regions) while, on the other hand, the assay is still functional, a combination that has proven difficult to fulfill.        
In many cases heat denaturation is not feasible. One example is when using an analytical system of a type exemplified by the Gyrolab™ system (Gyros AB, Uppsala, Sweden) where assays are performed in microfluidic structures provided in a spinnable compact disc (CD). Firstly, the heat treatment of the sample would have to be performed outside the CD and the instrument as there is no heating mechanism available therein. Secondly, it is likely that intra-CD heat treatment would destroy critical functions incorporated in the CD, potentially also generating protein aggregates which are incompatible with microfluidic-based assay principles. When heating is performed outside the CD, it is possible that protein particulates might be formed with the risk of clogging microstructures unless appropriate precautions are taken.
WO 2008/033073 A1 discloses a method of determining the total concentration of an analyte in a fluid sample, wherein at least part of the analyte is present as a complex with an analyte-binding species. The method comprises the steps of: a) subjecting the sample to conditions that reduce the binding affinity between analyte and analyte-binding species sufficiently to dissociate substantially any analyte complex and provide substantially all analyte in free form, b) subjecting the sample to conditions that restore the binding affinity between analyte and analyte-binding species, and c) immediately after the binding affinity has been restored, and before any substantial re-complexing of the analyte has taken place, determining the concentration of free analyte in the sample. In one embodiment, the method is performed in a flow system using label-free detection, such as surface plasmon resonance (SPR).
WO 2009/022001 A1 discloses a method based on surface plasmon resonance for detection of anti-drug antibodies (ADAs) against a therapeutic drug. Drug interference in the presence of drug in the patient sample to be analysed is overcome by acidifying the sample (pH 2.5 or 3), and then neutralizing the sample before analysis.
It is an object of the present invention to provide a method for quantifying total analyte in a sample, including analyte in complexed form, which is based on complex dissociation by acid treatment and which is generally functional for a variety of analytes and capturing agents, especially antibodies.